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1.
Nature ; 595(7866): 272-277, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34163067

RESUMO

Diet is a major factor that shapes the gut microbiome1, but the consequences of diet-induced changes in the microbiome for host pathophysiology remain poorly understood. We conducted a randomized human intervention study using a very-low-calorie diet (NCT01105143). Although metabolic health was improved, severe calorie restriction led to a decrease in bacterial abundance and restructuring of the gut microbiome. Transplantation of post-diet microbiota to mice decreased their body weight and adiposity relative to mice that received pre-diet microbiota. Weight loss was associated with impaired nutrient absorption and enrichment in Clostridioides difficile, which was consistent with a decrease in bile acids and was sufficient to replicate metabolic phenotypes in mice in a toxin-dependent manner. These results emphasize the importance of diet-microbiome interactions in modulating host energy balance and the need to understand the role of diet in the interplay between pathogenic and beneficial symbionts.


Assuntos
Bactérias/isolamento & purificação , Bactérias/metabolismo , Restrição Calórica , Dieta Redutora , Microbioma Gastrointestinal/fisiologia , Adiposidade , Animais , Bactérias/crescimento & desenvolvimento , Bactérias/patogenicidade , Toxinas Bacterianas/metabolismo , Ácidos e Sais Biliares/metabolismo , Peso Corporal , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/isolamento & purificação , Clostridioides difficile/metabolismo , Metabolismo Energético , Humanos , Absorção Intestinal , Masculino , Camundongos , Nutrientes/metabolismo , Simbiose , Perda de Peso
2.
Mol Cell ; 81(15): 3160-3170.e9, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34174184

RESUMO

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.


Assuntos
Toxinas Bacterianas/metabolismo , Guanosina Pentafosfato/metabolismo , Ligases/metabolismo , RNA de Transferência/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacologia , Bacilos Gram-Positivos Asporogênicos/química , Bacilos Gram-Positivos Asporogênicos/metabolismo , Guanosina Pentafosfato/química , Ligases/química , Ligases/genética , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/fisiologia , Inibidores da Síntese de Proteínas/farmacologia , Pirofosfatases , Ribossomos/metabolismo
3.
Nat Commun ; 12(1): 3748, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145250

RESUMO

C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for TcdB, but its pathophysiological relevance and the molecular details that govern recognition remain unknown. Here, we determine the cryo-EM structure of a TcdB-CSPG4 complex, revealing a unique binding site spatially composed of multiple discontinuous regions across TcdB. Mutations that selectively disrupt CSPG4 binding reduce TcdB toxicity in mice, while CSPG4-knockout mice show reduced damage to colonic tissues during C. difficile infections. We further show that bezlotoxumab, the only FDA approved anti-TcdB antibody, blocks CSPG4 binding via an allosteric mechanism, but it displays low neutralizing potency on many TcdB variants from epidemic hypervirulent strains due to sequence variations in its epitopes. In contrast, a CSPG4-mimicking decoy neutralizes major TcdB variants, suggesting a strategy to develop broad-spectrum therapeutics against TcdB.


Assuntos
Antígenos/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/patogenicidade , Enterocolite Pseudomembranosa/patologia , Proteoglicanas/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígenos/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Sítios de Ligação/fisiologia , Anticorpos Amplamente Neutralizantes/farmacologia , Microscopia Crioeletrônica , Enterocolite Pseudomembranosa/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Proteoglicanas/genética
4.
Commun Biol ; 4(1): 777, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162996

RESUMO

Over 100 kinds of proteins are expressed as glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) on the cell surface in mammalian cells. GPI-APs possess unique properties in terms of their intracellular trafficking and association with lipid rafts. Although it is clear that GPI-APs play critical roles in various biological phenomena, it is poorly understood how the GPI moiety contributes to these mechanisms. More than 30 genes are involved in the correct biosynthesis of GPI-APs. We here constructed a cell library in which 32 genes involved in GPI biosynthesis were knocked out in human embryonic kidney 293 cells. Using the cell library, the surface expression and sensitivity to phosphatidylinositol-specific phospholipase C of GPI-APs were analyzed. Furthermore, we identified structural motifs of GPIs that are recognized by a GPI-binding toxin, aerolysin. The cell-based GPI-knockout library could be applied not only to basic researches, but also to applications and methodologies related to GPI-APs.


Assuntos
Proteínas Ligadas por GPI/fisiologia , Glicosilfosfatidilinositóis/biossíntese , Toxinas Bacterianas/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Manosiltransferases/genética , Manosiltransferases/fisiologia , Proteínas Citotóxicas Formadoras de Poros/metabolismo
5.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073004

RESUMO

Type II toxin-antitoxin (TA) systems are genetic elements usually encoding two proteins: a stable toxin and an antitoxin, which binds the toxin and neutralizes its toxic effect. The disturbance in the intracellular toxin and antitoxin ratio typically leads to inhibition of bacterial growth or bacterial cell death. Despite the fact that TA modules are widespread in bacteria and archaea, the biological role of these systems is ambiguous. Nevertheless, a number of studies suggests that the TA modules are engaged in such important processes as biofilm formation, stress response or virulence and maintenance of mobile genetic elements. The Dickeya dadantii 3937 strain serves as a model for pathogens causing the soft-rot disease in a wide range of angiosperm plants. Until now, several chromosome-encoded type II TA systems were identified in silico in the genome of this economically important bacterium, however so far only one of them was experimentally validated. In this study, we investigated three putative type II TA systems in D. dadantii 3937: ccdAB2Dda, phd-docDda and dhiTA, which represents a novel toxin/antitoxin superfamily. We provide an experimental proof for their functionality in vivo both in D. dadantii and Escherichia coli. Finally, we examined the prevalence of those systems across the Pectobacteriaceae family by a phylogenetic analysis.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Dickeya , Doenças das Plantas/microbiologia , Sistemas Toxina-Antitoxina , Dickeya/genética , Dickeya/metabolismo , Dickeya/patogenicidade , Regulação Bacteriana da Expressão Gênica , Virulência
6.
Int J Biol Macromol ; 184: 497-508, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34126152

RESUMO

Anaplasma phagocytophilum is an obligate intracellular bacterium and a common tick-borne infectious pathogen that can cause human granulocytic anaplasmosis (HGA). Effector proteins play an important role in the pathogenic mechanism of A. phagocytophilum, but the specifics of the disease mechanism are unclear. We studied the effector protein AptA (A. phagocytophilum toxin A) using yeast two hybrid assays to screen its interacting protein proteasome assembly chaperone 3 (PSMG3, PAC3), and identified new mechanisms for the pathogenicity of A. phagocytophilum in HEK293T cells. After AptA enters the host cell, it interacts with PSMG3 to enhance the activity of the proteasome, causing ubiquitination and autophagy in the host cell and thereby increasing cross-talk between the ubiquitination-proteasome system (UPS) and autophagy. AptA also reduces the apoptotic efficiency of the host cells. These results offer new clues as to the pathogenic mechanism of A. phagocytophilum and support the hypothesis that AptA interacts with host PSMG3.


Assuntos
Anaplasma phagocytophilum/patogenicidade , Toxinas Bacterianas/metabolismo , Chaperonas Moleculares/metabolismo , Anaplasma phagocytophilum/metabolismo , Autofagia , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinação
7.
FASEB J ; 35(7): e21647, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34165206

RESUMO

The Cytotoxic Necrotizing Factor Y (CNFY) is produced by the gram-negative, enteric pathogen Yersinia pseudotuberculosis. The bacterial toxin belongs to a family of deamidases, which constitutively activate Rho GTPases, thereby balancing inflammatory processes. We identified heparan sulfate proteoglycans as essential host cell factors for intoxication with CNFY. Using flow cytometry, microscopy, knockout cell lines, pulsed electron-electron double resonance, and bio-layer interferometry, we studied the role of glucosaminoglycans in the intoxication process of CNFY. Especially the C-terminal part of CNFY, which encompasses the catalytic activity, binds with high affinity to heparan sulfates. CNFY binding with the N-terminal domain to a hypothetical protein receptor may support the interaction between the C-terminal domain and heparan sulfates, which seems sterically hindered in the full toxin. A second conformational change occurs by acidification of the endosome, probably allowing insertion of the hydrophobic regions of the toxin into the endosomal membrane. Our findings suggest that heparan sulfates play a major role for intoxication within the endosome, rather than being relevant for an interaction at the cell surface.


Assuntos
Toxinas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Linfócitos/metabolismo , Proteínas Recombinantes/metabolismo , Yersinia pseudotuberculosis/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Células HeLa , Humanos , Conformação Proteica , Proteínas Recombinantes/genética
8.
J Biol Chem ; 296: 100777, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33992647

RESUMO

Opportunistic bacteria strategically dampen their virulence to allow them to survive and propagate in hosts. However, the molecular mechanisms underlying virulence control are not clearly understood. Here, we found that the opportunistic pathogen Vibrio vulnificus biotype 3, which caused an outbreak of severe wound and intestinal infections associated with farmed tilapia, secretes significantly less virulent multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, which is the most critical virulence factor in other clinical Vibrio strains. The biotype 3 MARTX toxin contains a cysteine protease domain (CPD) evolutionarily retaining a unique autocleavage site and a distinct ß-flap region. CPD autoproteolytic activity is attenuated following its autocleavage because of the ß-flap region. This ß-flap blocks the active site, disabling further autoproteolytic processing and release of the modularly structured effector domains within the toxin. Expression of this altered CPD consequently results in attenuated release of effectors by the toxin and significantly reduces the virulence of V. vulnificus biotype 3 in cells and in mice. Bioinformatic analysis revealed that this virulence mechanism is shared in all biotype 3 strains. Thus, these data provide new insights into the mechanisms by which opportunistic bacteria persist in an environmental reservoir, prolonging the potential to cause outbreaks.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Vibrioses/metabolismo , Vibrio vulnificus/patogenicidade , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Modelos Moleculares , Vibrio vulnificus/fisiologia , Fatores de Virulência/química
9.
Appl Environ Microbiol ; 87(14): e0312120, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-33962981

RESUMO

Enterotoxigenic Escherichia coli (ETEC) and Shiga toxin-producing E. coli (STEC) strains are the causative agents of severe foodborne diseases in both humans and animals. In this study, porcine pathogenic E. coli strains (n = 277) as well as porcine commensal strains (n = 188) were tested for their susceptibilities to 34 bacteriocin monoproducers to identify the most suitable bacteriocin types inhibiting porcine pathogens. Under in vitro conditions, the set of pathogenic E. coli strains was found to be significantly more susceptible to the majority of tested bacteriocins than commensal E. coli. Based on the production of bacteriocins with specific activity against pathogens, three potentially probiotic commensal E. coli strains of human origin were selected. These strains were found to be able to outcompete ETEC strains expressing F4 or F18 fimbriae in liquid culture and also decreased the severity and duration of diarrhea in piglets during experimental ETEC infection as well as pathogen numbers on the last day of in vivo experimentation. While the extents of the probiotic effect were different for each strain, the cocktail of all three strains showed the most pronounced beneficial effects, suggesting synergy between the tested E. coli strains. IMPORTANCE Increasing levels of antibiotic resistance among bacteria also increase the need for alternatives to conventional antibiotic treatment. Pathogenic Escherichia coli represents a major diarrheic infectious agent of piglets in their postweaning period; however, available measures to control these infections are limited. This study describes three novel E. coli strains producing antimicrobial compounds (bacteriocins) that actively inhibit a majority of toxigenic E. coli strains. The beneficial effect of three potentially probiotic E. coli strains was demonstrated under both in vitro and in vivo conditions. The novel probiotic candidates may be used as prophylaxis during piglets' postweaning period to overcome common infections caused by E. coli.


Assuntos
Toxinas Bacterianas , Bacteriocinas/uso terapêutico , Infecções por Escherichia coli/prevenção & controle , Escherichia coli , Probióticos/uso terapêutico , Doenças dos Suínos/prevenção & controle , Animais , Toxinas Bacterianas/metabolismo , Bacteriocinas/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Fezes/microbiologia , Suínos , Doenças dos Suínos/microbiologia , Fatores de Virulência/genética
10.
Toxins (Basel) ; 13(5)2021 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946807

RESUMO

The fascinating world of lichens draws the attention of the researchers because of the numerous properties of lichens used traditionally and, in modern times, as a raw material for medicines and in the perfumery industry, for food and spices, for fodder, as dyes, and for other various purposes all over the world. However, lichens being widespread symbiotic entities between fungi and photosynthetic partners may acquire toxic features due to either the fungi, algae, or cyano-procaryotes producing toxins. By this way, several common lichens acquire toxic features. In this survey, recent data about the ecology, phytogenetics, and biology of some lichens with respect to the associated toxin-producing cyanoprokaryotes in different habitats around the world are discussed. Special attention is paid to the common toxins, called microcystin and nodularin, produced mainly by the Nostoc species. The effective application of a series of modern research methods to approach the issue of lichen toxicity as contributed by the cyanophotobiont partner is emphasized.


Assuntos
Toxinas Bacterianas/metabolismo , Cianobactérias/metabolismo , Ecotoxicologia , Líquens/metabolismo , Nostoc/metabolismo
11.
Microb Pathog ; 156: 104820, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33932546

RESUMO

Epsilon toxin (Etx) is an enormously potent pore-forming toxin and a category B biological agent. Etx is the main virulence determinant of Clostridiumperfringens types B and D toxin. It has a cytotoxic effect on distal and collecting kidney tubules. Also, Etx crosses the blood-brain barrier, binds to myelin structures, and destroys oligodendrocytes. The main purpose of this study was to investigate the toxic effects of Etx on human blood lymphocytes, which we examined for the first time for the genetic toxicity of this bacterial toxin. In this study, after taking blood and dividing into nine groups and putting in contact with different dilutions of Etx (1,5,10,25,50,100 and 200 µM), methotrexate (750 µM), and normal saline by Cytokinesis blocked micronucleus (CBMN) assay, we looked at genetic toxicity and the level of oxidative stress created in the under study lymphocytes. The results of this study showed that Etx has significant oxidative stress effects on human lymphocytes at doses above 25 µM, and also this bacterial toxin significantly increases the number of micronuclei formed in lymphocytes. The results of this study indicate that Etx has toxic effects it is genetic and interferes with cell division processes. Thus, human lymphocytes can be used extensively in future studies on Etx.


Assuntos
Toxinas Bacterianas , Clostridium perfringens , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Barreira Hematoencefálica/metabolismo , Clostridium perfringens/metabolismo , Humanos , Linfócitos/metabolismo , Estresse Oxidativo
12.
PLoS Pathog ; 17(4): e1009504, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33914853

RESUMO

Pathogens have evolved smart strategies to invade hosts and hijack their immune responses. One such strategy is the targeting of the host RhoGTPases by toxins or virulence factors to hijack the cytoskeleton dynamic and immune processes. In response to this microbial attack, the host has evolved an elegant strategy to monitor the function of virulence factors and toxins by sensing the abnormal activity of RhoGTPases. This innate immune strategy of sensing bacterial effector targeting RhoGTPase appears to be a bona fide example of effector-triggered immunity (ETI). Here, we review recently discovered mechanisms by which the host can sense the activity of these toxins through NOD and NOD-like receptors (NLRs).


Assuntos
Bactérias/imunologia , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas NLR/metabolismo , Transdução de Sinais , Fatores de Virulência/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Toxinas Bacterianas/metabolismo , Citoesqueleto/imunologia , Imunidade Inata , Pirina/metabolismo
13.
Nature ; 593(7858): 261-265, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33911281

RESUMO

Several enteric pathogens can gain specific metabolic advantages over other members of the microbiota by inducing host pathology and inflammation. The pathogen Clostridium difficile is responsible for a toxin-mediated colitis that causes 450,000 infections and 15,000 deaths in the United States each year1; however, the molecular mechanisms by which C. difficile benefits from this pathology remain unclear. To understand how the metabolism of C. difficile adapts to the inflammatory conditions that its toxins induce, here we use RNA sequencing to define, in a mouse model, the metabolic states of wild-type C. difficile and of an isogenic mutant that lacks toxins. By combining bacterial and mouse genetics, we demonstrate that C. difficile uses sorbitol derived from both diet and host. Host-derived sorbitol is produced by the enzyme aldose reductase, which is expressed by diverse immune cells and is upregulated during inflammation-including during toxin-mediated disease induced by C. difficile. This work highlights a mechanism by which C. difficile can use a host-derived nutrient that is generated during toxin-induced disease by an enzyme that has not previously been associated with infection.


Assuntos
Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidade , Infecções por Clostridium/metabolismo , Infecções por Clostridium/microbiologia , Interações Hospedeiro-Patógeno , Sorbitol/metabolismo , Aldeído Redutase/metabolismo , Animais , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Clostridioides difficile/genética , Infecções por Clostridium/enzimologia , Colite/enzimologia , Colite/metabolismo , Colite/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação
14.
Toxins (Basel) ; 13(4)2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33921797

RESUMO

Binary toxin (Bin toxin), BinA and BinB, produced by Lysinibacillus sphaericus has been used as a mosquito-control agent due to its high toxicity against the mosquito larvae. The crystal structures of Bin toxin and non-insecticidal but cytotoxic parasporin-2 toxin share some common structural features with those of the aerolysin-like toxin family, thus suggesting a common mechanism of pore formation of these toxins. Here we explored the possible cytotoxicity of Bin proteins (BinA, BinB and BinA + BinB) against Hs68 and HepG2 cell lines. The cytotoxicity of Bin proteins was evaluated using the trypan blue exclusion assay, MTT assay, morphological analysis and LDH efflux assay. The intracellular localization of Bin toxin in HepG2 cells was assessed by confocal laser scanning microscope. HepG2 cells treated with BinA and BinB (50 µg/mL) showed modified cell morphological features and reduced cell viability. Bin toxin showed no toxicity against Hs68 cells. The EC50 values against HepG2 at 24 h were 24 ng/mL for PS2 and 46.56 and 39.72 µg/mL for BinA and BinB, respectively. The induction of apoptosis in treated HepG2 cells was confirmed by upregulation of caspase levels. The results indicated that BinB mediates the translocation of BinA in HepG2 cells and subsequently associates with mitochondria. The study supports the possible development of Bin toxin as either an anticancer agent or a selective delivery vehicle of anticancer agents to target mitochondria of human cancer cells in the future.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bacillaceae/metabolismo , Toxinas Bacterianas/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Antineoplásicos/metabolismo , Toxinas Bacterianas/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Caspases/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Receptores de Superfície Celular/metabolismo
15.
Toxins (Basel) ; 13(5)2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33925254

RESUMO

Tuberculosis (TB) is a contagious disease caused by Mycobacterium tuberculosis (M. tb). It is regarded as a major health threat all over the world, mainly because of its high mortality and drug-resistant nature. Toxin-antitoxin (TA) systems are modules ubiquitously found in prokaryotic organisms, and the well-studied MazEF systems (MazE means "what is it?" in Hebrew) are implicated in the formation of "persister cells" in the M. tb pathogen. Here, we report cocrystal structures of M. tb MazF-mt1 and -mt9, two important MazF members responsible for specific mRNA and tRNA cleavages, respectively, in complexes with truncated forms of their cognate antitoxin peptides. These peptides bind to the toxins with comparable affinities to their full-length antitoxins, which would reduce the RNA-cleavage capacities of the toxins in vitro. After structural analysis of the binding modes, we systemically tested the influence of the substitutions of individual residues in the truncated MazE-mt9 peptide on its affinity. This study provides structural insight into the binding modes and the inhibition mechanisms between the MazE/F-mt TA pairs. More importantly, it contributes to the future design of peptide-based antimicrobial agents against TB and potentially relieves the drug-resistance problems by targeting novel M. tb proteins.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Endorribonucleases/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Sítios de Ligação , Endorribonucleases/química , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo
16.
BMC Infect Dis ; 21(1): 372, 2021 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-33882854

RESUMO

BACKGROUND: Some Staphylococcus aureus strains produce Panton-Valentine leukocidin (PVL), a bi-component pore-forming toxin, which causes leukocyte lysis and tissue necrosis. Currently, there is very limited information on the molecular epidemiology of PVL-encoding S. aureus strains in Iran. This study aimed to determine the molecular epidemiology and genetic background of PVL-positive S. aureus clinical strains isolated from Iranian patients. METHODS: A total of 28 PVL-positive S. aureus strains were detected from 600 S. aureus isolates between February 2015 and March 2018 from different hospitals in Tehran, Iran. Antimicrobial susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Molecular genotyping was performed using SCCmec and accessory gene regulator (agr) typing, PVL haplotyping, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). RESULTS: The highest antibiotic resistance rate was found to be against erythromycin (57.1%), followed by ciprofloxacin (42.8%) and clindamycin (35.7%). Moreover, 19 (67.9%) out of 28 S. aureus isolates were identified as MRSA, including CA-MRSA (14/19, 73.7%) and HA-MRSA (5/19, 26.3%). SCCmec type IVa was detected as the predominant type (10/19, 52.6%), followed by type III (5/19, 26.3%) and type V (4/19, 21.1%). The agr type I was identified as the most common type (14/28, 50%), and H and R haplotype groups were observed at frequencies of 67.9 and 32.1%, respectively. Among H variants, the predominant variant was H2 (78/9%). The isolates encompassed 21 different sequence types (STs), including 16 new STs (ST5147 to ST5162). Based on eBURST analysis, the isolates were clustered into five CCs, including CC30, CC22, CC1, CC8, and CC5 (ST5160), and nine singletons. PFGE typing showed that 24 isolates were clustered into A (4 pulsotypes), B (9 pulsotypes), and C (11 pulsotypes) clusters. CONCLUSIONS: A high prevalence of PVL-positive CA-MRSA strains was detected in Iran. The majority of PVL-positive isolates were of H (mostly H2) variant, while R variant was harbored by 100% of PVL-positive MRSA strains. Also, CC8, CC22, and CC30 were identified as the dominant clones among PVL-encoding S. aureus strains. This study promotes a better understanding of the molecular epidemiology and evolution of PVL-positive S. aureus strains in Iran.


Assuntos
Toxinas Bacterianas/genética , Exotoxinas/genética , Haplótipos , Leucocidinas/genética , Staphylococcus aureus Resistente à Meticilina/genética , Polimorfismo de Nucleotídeo Único , Infecções Estafilocócicas/epidemiologia , Antibacterianos/uso terapêutico , Toxinas Bacterianas/metabolismo , Farmacorresistência Bacteriana Múltipla/genética , Eletroforese em Gel de Campo Pulsado , Exotoxinas/metabolismo , Genômica , Humanos , Irã (Geográfico)/epidemiologia , Leucocidinas/metabolismo , Masculino , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Filogenia , Prevalência , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia
17.
J Med Microbiol ; 70(4)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33830907

RESUMO

Introduction. Cholix toxin (ChxA) is an ADP-ribosylating exotoxin produced by Vibrio cholerae. However, to date, there is no quantitative assay available for ChxA, which makes it difficult to detect and estimate the level of ChxA produced by V. cholerae.Hypothesis/Gap Statement. It is important to develop a reliable and specific quantitative assay to measure the production level of ChxA, which will help us to understand the role of ChxA in V. cholerae pathogenesis.Aim. The aim of this study was to develop a bead-based sandwich ELISA (bead-ELISA) for the quantification of ChxA and to evaluate the importance of ChxA in the pathogenesis of V. cholerae infection.Methodology. Anti-rChxA was raised in New Zealand white rabbits, and Fab-horse radish peroxidase conjugate was prepared by the maleimide method to use in the bead-ELISA. This anti-ChxA bead-ELISA was applied to quantify the ChxA produced by various V. cholerae strains. The production of ChxA was examined in different growth media such as alkaline peptone water (APW), Luria-Bertani broth and AKI. Finally, the assay was evaluated using a mouse lethality assay with representative V. cholerae strains categorized as low to high ChxA-producers based on anti-ChxA bead-ELISA.Results. A sensitive bead-ELISA assay, which can quantify from 0.6 to 60 ng ml-1 of ChxA, was developed. ChxA was mostly detected in the extracellular cell-free supernatant and its production level varied from 1.2 ng ml-1 to 1.6 µg ml-1. The highest ChxA production was observed when V. cholerae strains were cultured in LB broth, but not in APW or AKI medium. The ChxA-producer V. cholerae strains showed 20-80 % lethality and only the high ChxA II-producer was statistically more lethal than a non-ChxA-producer, in the mice model assay. ChxA I and II production levels were not well correlated with mice lethality, and this could be due to the heterogeneity of the strains tested.Conclusion. ChxA I to III was produced mostly extracellularly at various levels depending on strains and culture conditions. The bead-ELISA developed in this study is useful for the detection and quantification of ChxA in V. cholerae strains.


Assuntos
Fatores de Ribosilação do ADP/análise , Toxinas Bacterianas/análise , Ensaio de Imunoadsorção Enzimática , Vibrio cholerae/patogenicidade , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/imunologia , Fatores de Ribosilação do ADP/metabolismo , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/metabolismo , Cólera/microbiologia , Cólera/mortalidade , Meios de Cultivo Condicionados/química , Soros Imunes/imunologia , Imunoglobulina G/imunologia , Camundongos , Coelhos , Sensibilidade e Especificidade , Taxa de Sobrevida , Vibrio cholerae/genética , Vibrio cholerae/metabolismo
18.
Int J Mol Sci ; 22(6)2021 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805767

RESUMO

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell's cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Clostridioides difficile/patogenicidade , Infecção Hospitalar/tratamento farmacológico , Enterocolite Pseudomembranosa/tratamento farmacológico , Enterotoxinas/antagonistas & inibidores , ADP-Ribosilação/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/deficiência , Actinas/genética , Antibacterianos/uso terapêutico , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Sítios de Ligação , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Infecção Hospitalar/metabolismo , Infecção Hospitalar/microbiologia , Infecção Hospitalar/patologia , Endocitose/efeitos dos fármacos , Enterocolite Pseudomembranosa/metabolismo , Enterocolite Pseudomembranosa/microbiologia , Enterocolite Pseudomembranosa/patologia , Enterotoxinas/química , Enterotoxinas/genética , Enterotoxinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/ultraestrutura , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína
19.
Toxins (Basel) ; 13(3)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33800135

RESUMO

Current strategies for glioma treatment are only partly effective because of the poor selectivity for tumoral cells. Hence, the necessity to identify novel approaches is urgent. Recent studies highlighted the effectiveness of the bacterial protein cytotoxic necrotizing factor 1 (CNF1) in reducing tumoral mass, increasing survival of glioma-bearing mice and protecting peritumoral neural tissue from dysfunction. However, native CNF1 needs to be delivered into the brain, because of its incapacity to cross the blood-brain barrier (BBB) per se, thus hampering its clinical translation. To allow a non-invasive administration of CNF1, we here developed a chimeric protein (CTX-CNF1) conjugating CNF1 with chlorotoxin (CTX), a peptide already employed in clinics due to its ability of passing the BBB and selectively binding glioma cells. After systemic administration, we found that CTX-CNF1 is able to target glioma cells and significantly prolong survival of glioma-bearing mice. Our data point out the potentiality of CTX-CNF1 as a novel effective tool to treat gliomas.


Assuntos
Antineoplásicos/farmacologia , Toxinas Bacterianas/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Proteínas de Escherichia coli/farmacologia , Glioma/tratamento farmacológico , Venenos de Escorpião/farmacologia , Animais , Antineoplásicos/metabolismo , Toxinas Bacterianas/metabolismo , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Proteínas de Escherichia coli/metabolismo , Glioma/metabolismo , Glioma/patologia , Humanos , Injeções Intravenosas , Camundongos Endogâmicos C57BL , Proteínas Recombinantes de Fusão/farmacologia , Venenos de Escorpião/metabolismo
20.
Toxins (Basel) ; 13(3)2021 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-33805744

RESUMO

Clostridium perfringens is a Gram-positive bacterium that possess seven toxinotypes (A, B, C, D, E, F, and G) that are responsible for the production of six major toxins, i.e., α, ß, ε, ι, CPE, and NetB. The aim of this study is to find out the occurrence of toxinotypes in buffalo and cattle of Punjab province in Pakistan and their corresponding toxin-encoding genes from the isolated toxinotypes. To accomplish this aim, six districts in Punjab province were selected (i.e., Lahore, Sahiwal, Cheecha Watni, Bhakkar, Dera Ghazi Khan, and Bahawalpur) and a total of 240 buffalo and 240 cattle were selected for the collection of samples. From isolation and molecular analysis (16S rRNA), it was observed that out of seven toxinotypes (A-G), two toxinotypes (A and D) were found at most, whereas other toxinotypes, i.e., B, C, E, F, and G, were not found. The most frequently occurring toxinotype was type A (buffalo: 149/240; cattle: 157/240) whereas type D (buffalo: 8/240 cattle: 7/240) was found to occur the least. Genes encoding toxinotypes A and D were cpa and etx, respectively, whereas genes encoding other toxinotypes were not observed. The occurrence of isolated toxinotypes was studied using response surface methodology, which suggested a considerable occurrence of the isolated toxinotypes (A and D) in both buffalo and cattle. Association between type A and type D was found to be significant among the isolated toxinotypes in both buffalo and cattle (p ≤ 0.05). Correlation was also found to be positive and significant between type A and type D. C. perfringens exhibits a range of toxinotypes that can be diagnosed via genotyping, which is more reliable than classical toxinotyping.


Assuntos
Toxinas Bacterianas/genética , Búfalos/microbiologia , Bovinos/microbiologia , Clostridium perfringens/genética , Perfilação da Expressão Gênica , Toxicogenética , Transcriptoma , Animais , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Clostridium perfringens/metabolismo , Regulação Bacteriana da Expressão Gênica , Paquistão , Ribotipagem
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